Partition processing methods and systems

ABSTRACT

The disclosure provides methods for separating and/or purifying one or more molecules released from one or more fluid compartments or partitions, such as one or more droplets. Molecules can be released from a fluid compartment(s) and bound to supports that can be isolated via any suitable method, including example methods described herein. The disclosure also provides devices that can aid in isolating supports bound to molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Pat. ApplicationNo. 62/119,930 filed Feb. 24, 2015 the full disclosure of which isincorporated herein by reference in its entirety for all purposes.

BACKGROUND

Nucleic acid sequencing technology has experienced rapid and massiveadvances over recent years. As compared to gel based separation methodswhere nested sets of terminated sequence extension products wereinterpreted visually by scientists, today’s sequencing technologiesproduce enormous amounts of sequence data, allow illumination of neverbefore sequenced genomes and genome regions, and provide throughput andcosts that allow the widespread adoption of sequencing into routinebiological research and diagnostics.

Droplet-based microfluidic techniques are becoming a popular method ofpreparing molecules for sequencing due to massive high-throughput, lowreagent cost, and ease of preparation. However, droplet chemistrygenerates a complex mixture of chemicals and biological products thatcan impact the outcome of downstream applications. Therefore, thereexists a need for improved methods of purifying molecules from droplets.

SUMMARY

An aspect of the disclosure provides a method for recovering targetmolecules from a plurality of droplets, comprising: a) providing aplurality of droplets having contents that comprise a plurality oftarget molecules; b) releasing the contents from the plurality ofdroplets to provide released target molecules: c) contacting thereleased target molecules with one or more supports, wherein the one ormore supports bind the released target molecules to provide bound targetmolecules, and wherein the one or more supports comprises at least onecomponent responsive to a magnetic force; and d) applying the magneticforce to the one or more supports.

In some cases, releasing the contents from the plurality of droplets toprovide released target molecules further comprises destabilizing theplurality of droplets, thereby releasing the contents from the pluralityof droplets. In some cases, the plurality of droplets comprise dropletsin an emulsion and releasing the contents from the plurality of dropletsto provide released target molecules further comprises contacting theemulsion with a destabilization agent that destabilizes the emulsion,thereby releasing the contents from the plurality of droplets. In somecases, the destabilization agent comprises perfluorooctanol.

In some cases, releasing the contents from the plurality of droplets toprovide released target molecules further comprises releasing thecontents from the plurality of droplets into a pooled mixture, whereinthe pooled mixture comprises the released target molecules.

In some cases, the contacting step further comprises contacting thereleased molecules with the one or more supports by providing the one ormore supports to the pooled mixture. In some cases, the contacting stepfurther comprises contacting the released target molecules with the oneor more supports in the presence of a chaotrope that aids in the one ormore supports binding the released target molecules. In some cases, thechaotrope comprises guanadine thiocyanate or guanidine hydrochloride.

In some cases, the method provides for, after applying the magneticforce, releasing the bound target molecules from the one or moresupports to provide re-released target molecules. In some cases,releasing is completed with the aid of an elution agent. The elutionagent may comprise one or more of the following, without limitation:water, Tris buffer, phosphate buffer, and sodium hydroxide. Moreover, insome cases, prior to releasing the bound target molecules from the oneor more supports, the method further comprises washing the one or moresupports in one or more wash cycles by contacting the one or moresupports with a washing agent. In some cases, the washing agentcomprises ethanol, isopropanol and/or acetone. In some cases, the methodcomprises, after releasing the bound target molecules from the one ormore supports, subjecting the re-released target molecules to a solidphase reversible immobilization process.

In some cases, the target molecules comprise target nucleic acidmolecules. In some cases, the method further comprises, after thecontacting step, determining sequences of the target nucleic acidmolecules. In some cases, the method comprises, after the contactingstep, appending one or more additional nucleotides to the target nucleicacid molecules to provide larger target nucleic acid molecules. In somecases, the method further comprises determining sequences of the largertarget nucleic acid molecules.

In some cases, the target molecules comprise one or more of a smallmolecule, a protein, or a peptide.

In some cases, the at least one component comprises a magnetic particle.

In some instances, the one or more supports are functionalized with asilanol that aids in the one or more supports binding the targetmolecules. In some cases, the one or more supports are functionalizedwith a carboxylate that aids in the one or more supports binding thetarget molecules.

Further, the contents of the plurality of droplets may comprise one ormore nucleic acid molecules. The contents may further comprise apolymerase. The contents may further comprise a primer. In some cases,the primer comprises a barcode sequence. Further, the primer maycomprise a random N-mer.

In some cases, the contents of the plurality of droplets furthercomprise one or more polymeric species. The polymeric species maycomprise polyacrylamide. The polyacrylamide may comprise a linearpolyacrylamide. In some cases, the polyacrylamide may comprise agarose.In some instances, the contents of the plurality of droplets may furthercomprise a reducing agent.

In some cases, the plurality of droplets comprises aqueous droplets.Further, the plurality of droplets may comprise at least about 1,000droplets, at least about 10,000 droplets, at least about 100,000droplets, at least about 1,000,000 droplets or at least about 10,000,000droplets.

In some aspects, the disclosure provides a method for purifying a targetmolecule, comprising: a) in a vessel, providing a support to a liquidmixture comprising contents of a destabilized droplet that comprise atarget molecule, wherein the support binds the target molecule toprovide a bound target molecule; b) immobilizing the support at a firstlocation of the vessel, thereby separating the support from the liquidmixture: c) removing the liquid mixture from the vessel; d) providing asuspension fluid to the vessel, thereby suspending the support in thesuspension fluid; and e) immobilizing the support at a second locationof the vessel, thereby separating the support from the suspension fluid,wherein the second location of the vessel is different than the firstlocation of the vessel.

In some cases, in a), the liquid mixture further comprises a chaotropethat aids in the support binding the target molecule. The chaotrope maycomprise guanadine thiocyanate or guanidine hydrochloride. In somecases, in a), the liquid mixture further comprises a polymeric species.The polymeric species may comprise polyacrylamide. Furthermore, thepolyacrylamide may comprise a linear polyacrylamide. In some cases, thepolymeric species may comprise agarose.

In some cases, in a), the droplet is an aqueous droplet. Further, insome cases, in a). the liquid mixture further comprises adestabilization agent capable of destabilizing an emulsion. Thedestabilization agent may comprise PFO. Moreover, in some cases, in a),the liquid mixture further comprises a primer. The primer may comprise abarcode sequence. In some cases, the primer comprises a random N-mer.Furthermore, in a), the liquid mixture may further comprise a reducingagent.

In some cases, the immobilizing step further comprises magneticallyimmobilizing the support at the first location of the vessel. In someexamples, the removing step further comprises removing the liquidmixture from the vessel via suction. In some cases, the removing stepfurther comprises removing the liquid mixture from the vessel viadecanting. In some cases, in d), the suspension fluid comprises ethanol,isopropanol or acetone.

In some cases, the second immobilizing step further comprisesmagnetically immobilizing the support at the second location of thevessel.

In some cases, the method further comprises, after e), releasing thebound target molecule from the support, to provide a released targetmolecule. In some cases, releasing is completed with the aid of anelution agent. The elution agent may comprise one or more of thefollowing, without limitation: water, Tris buffer, phosphate buffer, andsodium hydroxide.

In some cases, prior to releasing the bound target molecule from thesupport, the method can further comprise washing the support in one ormore wash cycles by contacting the support with a washing agent. Thewashing agent may comprise one or more of the following, withoutlimitation: ethanol, isopropanol, and acetone.

In some cases, after releasing the bound target molecule from thesupport, the method can further comprise subjecting the released targetmolecule to a solid phase reversible immobilization process. In somecases, the target molecule comprises a target nucleic acid molecule.Furthermore, after e), the method can further comprise determining asequence of the target nucleic acid molecule. In some cases, after e),the method may further comprise appending one or more additionalnucleotides to the target nucleic acid molecule to provide a largertarget nucleic acid molecule. In some cases, the method may furthercomprise determining a sequence of the larger target nucleic acidmolecule. The target molecule may comprise a small molecule, a protein,a peptide.

In some cases, the support comprises a magnetic material. In some cases,the support comprises a particle. Furthermore, the support may befunctionalized with a silanol that aids the support in binding thetarget molecule. In some cases, the support is functionalized with acarboxylate that aids the support in binding the target molecule.

In some cases, the vessel may be a tube, a well, a dish and/or acontainer.

Another aspect of the disclosure provides a method for purifying atarget molecule, comprising: a) in a vessel, providing a liquid mixturecomprising: a target molecule; a support configured to bind the targetmolecule; a fluorinated oil; and a chaotrope; b) binding the targetmolecule to the support to provide a bound target molecule, wherein thechaotrope aids in binding the target molecule to the support; and c)separating the support from the liquid mixture under conditions thatimmobilize the support to a surface of the vessel.

In some cases, the fluorinated oil comprises a fluorocarbon oil. Thefluorinated oil may comprise hexafluoropropylene epoxide or a polymerthereof. The liquid mixture may further comprise a fluorosurfactant. Insome cases, the chaotrope comprises guanadine thiocyanate or guanidinehydrochloride.

In some cases, the liquid mixture further comprises a polymeric species.The polymeric species may comprise polyacrylamide. The polyacrylamidemay comprise a linear polyacrylamide. In some cases, the polymericspecies may comprise agarose. The liquid mixture may further comprise adestabilization agent capable of destabilizing an emulsion. Thedestabilization agent may comprise perfluorooctanol. In some cases, theliquid mixture further comprises a primer. The primer may comprise abarcode sequence. The primer may comprise a random N-mer. Furthermore,the liquid mixture may further comprise a polymerase. The liquid mixturemay further comprise a reducing agent.

In some cases, the separating step further comprises separating thesupport from the liquid mixture under conditions that magneticallyimmobilize the support to the surface of the vessel. In some cases,after c), the method may further comprise releasing the bound targetmolecule from the support to provide a released target molecule. Thereleasing step may be completed with the aid of an elution agent. Theelution agent may comprise one or more of the following, withoutlimitation: water, Tris buffer, phosphate buffer, and sodium hydroxide.

The method may further comprise, prior to releasing the bound targetmolecule from the support, washing the support in one or more washcycles by contacting the magnetic support with a washing agent. Thewashing agent may comprise one or more of the following, withoutlimitation: ethanol, isopropanol, and acetone. Furthermore, afterreleasing the bound target molecule from the support, the method canfurther comprise subjecting the released target molecule to a solidphase reversible immobilization process. The target molecule maycomprise a target nucleic acid molecule. The method may furthercomprise, after the separating step, determining a sequence of thetarget nucleic acid molecule.

In some cases, after the separating step, the method may furthercomprise appending one or more additional nucleotides to the targetnucleic acid molecule to provide a larger target nucleic acid molecule.Furthermore, the method may comprise determining a sequence of thelarger target nucleic acid molecule. The target molecule comprises asmall molecule, a protein, a peptide.

In some cases, the support comprises a magnetic material. In some cases,the support comprises a particle. In some instances, the support isfunctionalized with a silanol that aids in binding the target moleculeto the support. In other instances, the support is functionalized with acarboxylate that aids in binding the target molecule to the support.

In some examples, the vessel may be a tube, a well, a dish and/or acontainer.

Another aspect of the disclosure provides for a method for sequencing anucleic acid library, comprising: a) generating a library of nucleicacid molecules, wherein the library comprises a plurality of dropletscomprising the nucleic acid molecules; b) destabilizing the plurality ofdroplets, thereby releasing the nucleic acid molecules from theplurality of droplets into a common pool; c) recovering the nucleic acidmolecules from the common pool to provide recovered nucleic acidmolecules, wherein recovering comprises: i) in the common pool,immobilizing the nucleic acid molecules to a plurality of supports; andii) isolating the plurality of supports; and d) determining sequences ofat least a subset of the recovered nucleic acid molecules.

In some cases, the nucleic acid molecules comprise a barcode sequence.In some cases, the nucleic acid molecules comprise a random N-mer.

In some instances, the plurality of droplets comprises droplets in anemulsion. Furthermore, the destabilizing step may comprise destabilizingthe emulsion.

In some cases, the plurality of supports comprises particles.

Furthermore, the generating step may comprise barcoding sample nucleicacid molecules in the plurality of droplets. Moreover, the immobilizingstep may comprise, binding the nucleic acid molecules to the pluralityof supports via one or more ionic interactions. Furthermore, theisolating step of c) may comprise subjecting the plurality of supportsto one or more cycles of magnetic separation. The isolating step of c)may further comprise subjecting the plurality of supports to one or morecycles of centrifugation. In some cases, the method further comprises,prior to d), releasing the recovered nucleic acid molecules from theplurality of supports. In some cases, the method can further compriseappending one or more additional nucleotides to each of the recoverednucleic acid molecules to provide larger nucleic acid molecules.Furthermore, in d), determining sequences of at least the subset of therecovered nucleic acid molecules may comprise determining sequences ofat least a subset of larger nucleic acid molecules.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1G provide a schematic illustration of an example method forbarcoding and amplification of nucleic acid fragments;

FIG. 2 illustrates an example workflow for performing generating nucleicacid molecules for sequencing as described in Example 1;

FIGS. 3A-3F illustrate an example method for recovering target moleculesfrom droplets in an emulsion as described in Example 2;

FIG. 4A graphically illustrates product yields obtained from experimentsdescribed in Example 3; FIG. 4B graphically illustrates sequencing dataobtained from experiments described in Example 3;

FIG. 5 graphically illustrates product yields obtained from experimentsdescribed in Example 4;

FIGS. 6A-6C schematically illustrate an example magnetic separationdevice; FIG. 6D schematically illustrates an example use of the examplemagnetic separation device of FIGS. 6A-6C; and

FIG. 7A schematically illustrates an example magnetic separation deviceand its use; FIGS. 7B and 7C provide additional view of the examplemagnetic separation device of FIG. 7A.

FIGS. 8A and 8B schematically illustrate an alternative example magneticseparation device.

FIGS. 9A-9C schematically illustrate another alternative examplemagnetic separation device.

DETAILED DESCRIPTION

Described herein are methods for isolating the content(s) of apartition, such as a droplet. The methods described herein are usefulfor isolating target molecule(s) from a mixture that can comprise thecontents of one or more partitions. Moreover, the methods describedherein may also be useful in the generation of nucleic acid librariesfor sequencing. For example, a nucleic acid molecule library (e.g., asequencing library) may be generated in a plurality of droplets in anemulsion. The contents of the droplets can be released from thedroplets, by, for example, destabilizing or breaking the emulsion suchthat the contents are pooled in a common mixture that includes thenucleic acid molecule library. The nucleic acid molecules in the commonmixture can be bound to a plurality of supports. Upon isolating thesupports, the nucleic acid molecules can be isolated from the mixture.The nucleic acid molecules can then be washed and the supports isolatedin one or more additional cycles, followed by elution of the nucleicacid molecules from the supports. The released nucleic acid moleculescan then be further processed and analyzed, such as, for example,sequenced in a nucleic acid sequencing reaction.

Droplets and Emulsions

In one aspect, the methods herein provide for recovering targetmolecules from a plurality of droplets. In some examples, the methodsprovide for providing a plurality of droplets having contents thatcomprise a plurality of target molecules. As used herein, dropletsgenerally refer to small globules of one liquid suspended in a secondliquid. Droplets can be formed when two or more immiscible liquids aremixed such as, for example, water and oil. An example of a mixturecomprising two or more immiscible liquids is an emulsion, such as awater-in-oil emulsion. The first liquid, which is dispersed in globules,can be referred to as a discontinuous phase, whereas the second liquid,in which the globules are dispersed, can be referred to as a continuousphase or dispersion medium. In some examples, the continuous phase canbe a hydrophobic fluid, such as an oil, and the discontinuous phase canbe an aqueous phase solution. Such a mixture can be considered awater-in-oil emulsion, wherein aqueous droplets are dispersed in an oilcontinuous phase. In other cases, an emulsion may be an oil-in-wateremulsion. In such an emulsion, the discontinuous phase is a hydrophobicsolution (e.g., oil) and the continuous phase is an aqueous solution,wherein droplets of oil are dispersed in an aqueous phase. In someexamples, the emulsion may comprise a multiple emulsion. Multipleemulsions can comprise larger fluidic droplets that encompass one ormore smaller droplets (i.e., a droplet within a droplet). Multipleemulsions can contain one, two, three, four, or more nested fluidsgenerating increasingly complex droplets within droplets.

An oil of an emulsion may be selected based upon chemical properties,such as, among others molecular structure, content, solvating strength,viscosity, boiling point, thermal expansion coefficient, oil-in-watersolubility, water-in-oil solubility, dielectric constant, polarity,water-in-oil surface tension, and/or oil-in-water surface tension.Examples of oils useful in an emulsion (e.g.., a water-in-oil emulsion)include, without limitation, fluorinated oils, non-fluorinated oils,alkanes (e.g., hexane, decane, octane, and the like), mineral oils,plant oils, vegetable oils, comestible oils, mineral oil, oleic acid,embryo-tested mineral oil, light mineral oil, heavy mineral oil, PCRmineral oil, AS4 silicone oil, AS 100 silicone oil, AR20 silicone oil,AR 200 silicone oil. AR 1000 silicone oil, AP 100 silicone oil, AP 1000silicone oil, AP 150 silicone oil, AP 200 silicone oil, CR 200 Siliconeoil, DC 200 silicone oil, DC702 silicone oil. DC 710 silicone oil,octanol, decanol, acetophenone, perfluoro-oils, perfluorononane,perfluorodecane, perfluorodimethylcylcohexane,perfluoro-1-butanesulfonyl fluoride, perfluoro-1-octanesulfonylfluoride, perfluoro-1-octanesulfonyl fluoride,nonafluoro-1-butancsulfonyl chloride, nonafluoro-tert-butyl alcohol,perfluorodecanol, perfluorohexane, perfluorooctanol, perfluorodecene,perfluorohexene, perfluorooctene, fuel oil, halocarbon oil 28,halocarbon oil 700, hydrocarbon oil, glycerol, 3M Fluoriner™ fluids(FC-40, FC-43, FC-70, FC-72, FC-77, FC-84. FC-87, FC-3283), oilscomprising trifluoroacetic acid, oils comprising hexafluoroisopropanol,Krytox oils (e.g., oils comprising hexafluoropropylene epoxide and/orpolymers thereof), oil comprising polyhexafluoropropylene oxide and/orpolymers thereof, Krytox GPL oils, oils comprising perfluoropolyether,oils comprising perfluoroalkylether, oils comprisingperfluoropolyalkylether, Solvay Galden oils, oils including oils includehydrofluoroethers (e.g., HFE-7500, HFE-7100, HFE-7200, HFE-7600), oilscomprising perfluoroalkylamines (e.g., Fluorinert FC-3283 and FluorinertFC-40), soybean oil, castor oil, coconut oil, cedar oil, clove bud oil,fir oil, linseed oil, safflower oil, sunflower oil, almond seed oil,anise oil, clove oil, cottonseed oil, corn oil, croton oil, olive oil,palm oil, peanut oil, bay oil, borage oil, bergamot oil, cod liver oil,macadamia nut oil, camada oil, chamomile oil, citronella oil, eucalyptusoil, fennel oil, lavender oil, lemon oil, nutmeg oil orange oil,petitgrain oil, rose oil, tarragon oil, tung oil, basil oil, birch oil,black pepper oil, birch tar oil, carrot seed oil, cardamom oil, cassiaoil, sage oil, cognac oil, copaiba balsam oil, cypress oil, eucalyptusoil, dillweed oil, grape fruit oil, ginger oil, juniper oil, lavenderoil, lovage oil, majoram oil, mandarin oil, myrrh oil, neroli oil,olibanum oil, onion oil, paraffin oil, origanum oil, parsley oil,peppermint oil, pimenta leaf oil, sage oil, rosemary oil, rose oil,sandalwood oil, sassafras oil, spearmint oil, thyme oil, transformeroil, verbena oil, and rapeseed oil. In some examples, a water-in-oilemulsion may comprise one or more of the oils described herein, whereinaqueous droplets are dispersed in the oil(s).

An emulsion may further comprise a surfactant. The surfactant may be afluorosurfactant. Surfactants are known to stabilize droplets in acontinuous phase. Examples of fluorosurfactants useful for stabilizingdroplets are described in detail in U.S. Pat.Publication 2010-0105112,which is hereby incorporated by reference in its entirety. In someexamples, a water-in-oil emulsion may comprise one or more of the oilsdescribed herein having one or more surfactants (e.g.,fluorosurfactants), wherein aqueous droplets are dispersed in theoil(s).

Droplets may be formed by a variety of methods. Emulsion systems forcreating stable droplets in non-aqueous or oil continuous phases aredescribed in detail in, e.g., Published U.S. Pat. Publication No.2010-0105112. In some cases, microfluidic channel networks areparticularly suited for generating droplets as described herein.Examples of such microfluidic devices include those described in detailin Provisional U.S. Pat. Application No. 61/977,804. filed Apr. 4, 2014,the full disclosure of which is incorporated herein by reference in itsentirety for all purposes. Droplets may be formed with a regularperiodicity or may be formed with an irregular periodicity. In someaspects, the size and/or shape of the droplet may be determined by thesize and shape of a channel in which the droplet is formed.

In some examples, droplets may generally be generated by flowing anaqueous stream into a junction of two or more channels of a microfluidicsystem into which is also flowing a non-aqueous stream of fluid, e.g., afluorinated oil, such that aqueous droplets are created within theflowing stream non-aqueous fluid. The aqueous stream can include one ormore species such that upon droplet formation, the droplet contentscomprise aqueous interiors comprising the one or more species.Additional examples of such species are provided elsewhere herein. Therelative amount of species within a droplet may be adjusted bycontrolling a variety of different parameters of the system, including,for example, the concentration of species in the aqueous stream, theflow rate of the aqueous stream and/or the non-aqueous stream, and thelike.

Droplets may have overall volumes that are less than 1000 pL, less than900 pL, less than 800 pL, less than 700 pL, less than 600 pL, less than500 pL, less than 400pL, less than 300 pL, less than 200 pL, less than100pL. less than 50 pL, less than 20 pL, less than 10 pL, or even lessthan 1pL. Droplets may be monodisperse (i.e., substantially uniform insize) or polydisperse (i.e.. substantially non-uniform in size). Aplurality of droplets may be generated.

An emulsion may comprise a varied number of droplets depending upon theparticular emulsion. For example, an emulsion may comprise at least 10droplets, at least 50 droplets, at least 100 droplets, at least 500droplets, at least 1000 droplets, at least 5000 droplets, at least10,000 droplets, at least 50,000 droplets, at least 100,000 droplets, atleast 500,000 droplets, at least 1,000.000 droplets, at least 5,000,000droplets, at least 10,000,000 droplets, at least 50,000,000 droplets, atleast 100,000,000 droplets and upwards.

Contents of Droplets

Droplets can encapsulate one or more species, such as one or more targetmolecules and/or particles. Put another way, a droplet can be a discretepartition, isolating one or more target molecules and/or particles.Generally, a discontinuous phase can be selected to be compatible withthe target molecule(s) and/or particle(s) that are encapsulated. Forexample, a nucleic acid can be encapsulated in an aqueous droplet (e.g.,a buffer). A target molecule can generally refer to a species ofparticular interest in which further analysis or sequestration of thespecies is desired. For example a target molecule may be a targetnucleic acid molecule. A target nucleic acid molecule may be, forexample, single-stranded, partially single-stranded, partiallydouble-stranded, or double-stranded. A target nucleic acid molecule maybe any type of nucleic acid with non-limiting examples that includeoligonucleotides, nucleotides, DNA. RNA, peptide polynucleotides,complementary DNA (cDNA), double stranded DNA (dsDNA), single strandedDNA (ssDNA), plasmid DNA, cosmid DNA. chromosomal DNA, genomic DNA.viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA. tRNA,nRNA, siRNA, snRNA. snoRNA, scaRNA, microRNA, dsRNA, ribozyme,riboswitch and viral RNA, a locked nucleic acid (LNA) in whole or part,locked nucleic acid nucleotides and any other type of nucleic acidanalogue

Other non-limiting examples of target molecules include peptides,proteins, small molecules and the like. In some cases a plurality ofdroplets may comprise a plurality of target molecules, such as aplurality of target nucleic acid molecules. In such cases, the pluralityof droplets may be a nucleic acid library, wherein the droplets comprisenucleic acids for sequencing (e.g., a sequencing library).

A droplet may comprise a single target molecule or may comprise e aplurality of target molecules (e.g.. target molecules). In someexamples, one or more target molecules may be bound to a solid support.For examples, target molecules may be nucleic acids bound to a bead(e.g., polyacrylamide bead). In other examples, a target molecule may bea protein expressed on the surface of a biological cell. Targetmolecules may be the product of a chemical or biological reaction asdescribed elsewhere herein.

Droplets may comprise any suitable type and/or number of species thatcan include, but are not limited to, nucleic acids (DNA), proteins,small molecules, peptides, biological cells (e.g., mammalian cells,bacterial cells), and the like. The contents of droplets may alsocomprise one or more particles, such as, for example beads. A particlemay be any form of minute matter, natural or synthetic, that is smallenough in size to be encapsulated by a droplet. In further examples,droplets may encapsulate a combination of particles and other speciesdescribed herein. For example, a droplet may encapsulate a particlecoated or bound with one or more additional species, such as, forexample, a bead coated with nucleic acids.

In some examples, a droplet may comprise a particle such as bead,including gel beads and other types of beads. In particular, theseparticles may provide a surface to which reagents are releasablyattached, or a volume in which reagents are entrained or otherwisereleasably partitioned. These reagents may then be delivered inaccordance with a desired method, for example, in the controlleddelivery of reagents into droplets. A wide variety of different reagentsor reagent types may be associated with the particles, where one maydesire to deliver such reagents to a droplet. Non-limiting examples ofsuch reagents include, e.g., enzymes, polypeptides, antibodies orantibody fragments, labeling reagents, e.g., dyes, fluorophores,chromophores, etc., nucleic acids, polynucleotides, oligonucleotides,and any combination of two or more of the foregoing. In some cases, theparticles may provide a surface upon which to synthesize or attacholigonucleotide sequences. Various entities including oligonucleotides,barcode sequences, primers, crosslinkers and the like may be associatedwith the outer surface of a particle. In the case of porous particles,an entity may be associated with both the outer and inner surfaces of aparticle. The entities may be attached directly to the surface of aparticle (e.g., via a covalent bond, ionic bond, van der Waalsinteractions, etc.), may be attached to other oligonucleotide sequencesattached to the surface of a particle (e.g. adaptor or primers), may bediffused throughout the interior of a particle and/or may be combinedwith a particle in a partition (e.g. fluidic droplet). In some cases, anentity such as a cell or nucleic acid is encapsulated within a particle.Other entities including amplification reagents (e.g., PCR reagents,primers) may also be diffused throughout the particle orchemically-linked within the interior (e.g., via pores, covalentattachment to polymeric matrix) of a particle. Additional examples ofparticles that may be useful are described in U.S. Pat. Publication2014-0378345, the full disclosure of which is hereby incorporated byreference in its entirety for all purposes.

A particle may comprise natural and/or synthetic materials, includingnatural and synthetic polymers. Examples of natural polymers includeproteins and sugars such as deoxyribonucleic acid, rubber, cellulose,starch (e.g. amylose, amylopectin), proteins, enzymes, polysaccharides,silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) andcombinations (e.g., co-polymers) thereof. Particles may also be formedfrom materials other than polymers, including lipids, micelles,ceramics, glass-ceramics, material composites, metals, other inorganicmaterials, and others. In exemplary cases, the particle may comprisepolyacrylamide. The polyacrylamide particle may comprise linearpolyacrylamide (LPA). In other examples, the particle may compriseagarose.

In certain examples, reagents for one or more chemical or biologicalreaction can be encapsulated such that a droplet functions as a smallreaction chamber in which a chemical or biological reaction(s) may takeplace. In these examples, the discontinuous phase can be selected to becompatible with the desired chemical or biological reaction(s) (i.e.,can provide suitable conditions for the reaction to occur). In the caseof a chemical or biological reaction, a droplet may include suitablecomponents for the chemical or biological reaction to take place, suchas, for example an enzyme, reactants, any necessary cofactors, etc. Oncethe reaction(s) takes place, the droplet can also comprise anyproducts/by-products of the reaction(s). The biological reaction can beany number of enzymatic reactions that can be carried out in a droplet.

An example of a biological reaction that may take place in a dropletincludes a primer extension reaction that may be useful in a nucleicacid amplification reaction, such as, for example a polymerase chainreaction (PCR). In cases where a primer extension reaction takes placein a droplet, the droplet may comprise components for a primer extensionreaction (i.e., template nucleic acid, primers, a polymerase, dNTPs, andthe like). In some cases, the contents of the droplets may comprise apolymerase and/or any other enzyme for use in an amplification reaction.In other cases, the contents of the droplets may comprise a primer. Insome cases, the primer may comprise a barcode sequence. In some cases,the primers may comprise a random N-mer. Examples of barcoding nucleicacid molecules in droplets are described below. As described elsewhereherein, a target molecule can be a target nucleic acid molecule. Thetarget nucleic acid molecule may act as a template for an amplificationreaction. Additional examples of amplification reactions that may becompleted in droplets are provided by U.S. Pat. Application 14/316,383,filed Jun. 26, 2014, the full disclosure of which is incorporated hereinby reference in its entirety for all purposes.

An example of a chemical reaction that can be conducted within a dropletis the dissolution or degradation of a particle in the droplet, such asa bead. A degradable particle may comprise one or more species with alabile bond such that when the particle/species is exposed to theappropriate stimuli, the bond is broken and the particle degrades. Thelabile bond may be a chemical bond (e.g., covalent bond, ionic bond) ormay be another type of physical interaction (e.g., van der Waalsinteractions, dipole-dipole interactions, etc.). In some cases, acrosslinker used to generate a particle may comprise a labile bond. Uponexposure to the appropriate conditions, the labile bond is broken andthe particle is degraded. For example, a polyacrylamide gel particle maycomprise cystamine crosslinkers. Upon exposure of the particle to areducing agent, the disulfide bonds of the cystamine are broken and theparticle is degraded. Accordingly, a droplet may comprise a reducingagent that is capable of dissolving or degrading a particle having oneor more disulfide bonds. Moreover, in the case of a polymeric particle,a droplet may comprise polymeric by-products (e.g., polymeric species)of a degraded particle. For example, in the case of a polyacrylamideparticle, the droplet may comprise one or more polyacrylamide species,such as linear polyacrylamide (LPA), resulting from degradation of theparticle.

Particles may also be degradable, disruptable, or dissolvablespontaneously or upon exposure to one or more stimuli (e.g., temperaturechanges, pH changes, exposure to chemical species or phase, exposure tolight, reducing agent, etc.). In some cases, a particle may bedissolvable, such that material components of the particles aresolubilized when exposed to a particular chemical species or anenvironmental change, such as, for example, temperature, or pH. Forexample, a gel bead may be degraded or dissolved at elevated temperatureand/or in basic conditions. In some cases, a particle may be thermallydegradable such that when the bead is exposed to an appropriate changein temperature (e.g., heat), the particle degrades. Degradation ordissolution of a particle bound to a species (e.g., a nucleic acidspecies) may result in release of the species from the particle.

FIGS. 1A-1G show an example of an amplification reaction that can beperformed in a droplet and can be useful for generating a nucleic acidsequencing library in a plurality of droplets. In this example,oligonucleotides that include a barcode sequence are co-partitioned in.e.g., a droplet 102 in an emulsion, along with a sample nucleic acid 104(e.g., a target nucleic acid molecule). As noted elsewhere herein, theoligonucleotides 108 may be provided on a bead 106 that isco-partitioned with the sample nucleic acid 104, which oligonucleotidescan be releasable from the bead 106 (e.g., via degradation of one ormore labile bonds of the bead), as shown in FIG. 1A. Theoligonucleotides 108 include a barcode sequence 112, in addition to oneor more functional sequences, e.g., sequences 110, 114 and 116. Forexample, oligonucleotide 108 is shown as comprising barcode sequence112, as well as sequence 110 that may function as an attachment orimmobilization sequence for a given sequencing system, e.g.. a P5sequence used for attachment in flow cells of an Illumina Hiseq or Miseqsystem. As shown, the oligonucleotides also include a primer sequence116, which may include a random or targeted N-mer for primingreplication of portions of the sample nucleic acid 104. Also includedwithin oligonucleotide 108 is a sequence 114 which may provide asequencing priming region, such as a “read1” or R1 priming region, thatis used to prime polymerase mediated, template directed sequencing bysynthesis reactions in sequencing systems. In many cases, the barcodesequence 112, immobilization sequence 110 and R1 sequence 114 may becommon to all of the oligonucleotides attached to a given bead. Theprimer sequence 116 may vary for random N-mer primers, or may be commonto the oligonucleotides on a given bead for certain targetedapplications.

Based upon the presence of primer sequence 116, the oligonucleotides areable to prime the sample nucleic acid as shown in FIG. 1B, which allowsfor extension of the oligonucleotides 108 and 108 a using polymeraseenzymes and other extension reagents also co-portioned with the bead 106and sample nucleic acid 104. As shown in FIG. 1C, following extension ofthe oligonucleotides that, for random N-mer primers, would anneal tomultiple different regions of the sample nucleic acid 104; multipleoverlapping complements or fragments of the nucleic acid are created,e.g.. fragments 118 and 120. Although including sequence portions thatare complementary to portions of sample nucleic acid, e.g., sequences122 and 124. these constructs are generally referred to herein ascomprising fragments of the sample nucleic acid 104, having the attachedbarcode sequences. As can be appreciated, the replicated portions of thetemplate sequences as described above are often referred to herein as“fragments” of that template sequence. Notwithstanding the foregoing,however, the term “fragment” encompasses any representation of a portionof the originating nucleic acid sequence, e.g.. a template or samplenucleic acid, including those created by other mechanisms of providingportions of the template sequence, such as actual fragmentation of agiven molecule of sequence, e.g.. through enzymatic, chemical ormechanical fragmentation. In some cases, however, fragments of atemplate or sample nucleic acid sequence can denote replicated portionsof the underlying sequence or complements thereof.

The barcoded nucleic acid fragments may then be subjected tocharacterization. e.g., through sequence analysis, or they may befurther amplified in the process, as shown in FIG. 1D. For example,additional oligonucleotides, e.g., oligonucleotide 108 b, also releasedfrom bead 106, may prime the fragments 118 and 120. In particular,again, based upon the presence of the random N-mer primer 116 b inoligonucleotide 108 b (which in many cases may be different from otherrandom N-mers in a given droplet, e.g., primer sequence 116), theoligonucleotide anneals with the fragment 118, and is extended to createa complement 126 to at least a portion of fragment 118 which includessequence 128, that comprises a duplicate of a portion of the samplenucleic acid sequence. Extension of the oligonucleotide 108 b continuesuntil it has replicated through the oligonucleotide portion 108 offragment 118. As noted elsewhere herein, and as illustrated in FIG. 1D,the oligonucleotides may be configured to prompt a stop in thereplication by the polymerase at a desired point, e.g., afterreplicating through sequences 116 and 114 of oligonucleotide 108 that isincluded within fragment 118. This may be accomplished by differentmethods, including, for example, the incorporation of differentnucleotides and/or nucleotide analogues that are not capable of beingprocessed by the polymerase enzyme used. For example, this may includethe inclusion of uracil containing nucleotides within the sequenceregion 112 to prevent a non-uracil tolerant polymerase to ceasereplication of that region, As a result a fragment 126 is created thatincludes the full-length oligonucleotide 108 b at one end, including thebarcode sequence 112, the attachment sequence 110, the R1 primer region114, and the random N-mer sequence 116 b. At the other end of thesequence may be included the complement 116′ to the random N-iner of thefirst oligonucleotide 108, as well as a complement to all or a portionof the R1 sequence, shown as sequence 114′. The R1 sequence 114 and itscomplement 114′ are then able to hybridize together to form a partialhairpin structure 128. As can be appreciated because the random N-mersdiffer among different oligonucleotides, these sequences and theircomplements would not be expected to participate in hairpin formation,e.g., sequence 116′, which is the complement to random N-mer 116, wouldnot be expected to be complementary to random N-mer sequence 116 b. Thiswould not be the case for other applications, e.g., targeted primers,where the N-mers would be common among oligonucleotides within a givendroplet. By forming these partial hairpin structures, it allows for theremoval of first level duplicates of the sample sequence from furtherreplication, e.g., preventing iterative copying of copies. The partialhairpin structure also provides a useful structure for subsequentprocessing of the created fragments, e.g., fragment 126.

All of the fragments from multiple different droplets may then be pooled(e.g., by collecting droplets and destabilizing the emulsion asdescribed elsewhere herein) for sequencing on high throughputsequencers. Because each fragment is coded as to its droplet of origin,the sequence of that fragment may be attributed back to its origin basedupon the presence of the barcode.

As can be appreciated, the example amplification scheme depicted inFIGS. 1A-1G may be completed in any suitable type of partition,including non-droplet partitions, such as microcapsules, wells (e.g.,microwells), polymeric capsules, microreactors, micelles, etc.

Additional examples of amplification reactions that can be performed indroplets or other types of partitions, including amplification reactionsthat can be used to generate nucleic acid libraries for sequencing, areprovided in U.S. Provisional Pat. Application No. 62/102,420, filed Jan.12, 2015, which is incorporated herein by reference in its entirety forall purposes.

Releasing Contents of Droplets to Form a Pooled Mixture

Methods described herein provide for releasing the contents of a dropletor a plurality of droplets, such as, for example releasing one or moretarget molecules from a droplet or plurality of droplets. Generally, thecontents of a droplet or a plurality of droplets can contain one or moretarget molecules and it may be desirable to recover the targetmolecule(s). A target molecule or target molecules that have beenreleased from a droplet or plurality of droplets are herein referred toas a “released target molecule” or “released target molecules.”Releasing can encompass any method by which the contents of a dropletare liberated. Examples of releasing can include breaking the surface ofthe droplet, making the droplet porous such that the contents candiffuse out of the droplet, or any other method in which the contents ofthe droplet would be liberated.

In some cases, a droplet or a plurality of droplets can be destabilized(broken) to release the contents of the droplet(s) into a pooledmixture. For example, destabilizing a droplet or plurality of dropletsin an emulsion may comprise destabilizing the emulsion. The terms“destabilize,” “break,” “burst,” and “de-emulsify” may be usedinterchangeably herein. Methods of destabilizing droplets are known tothose of skill in the art. Briefly, droplets in an emulsion can be mixedwith a destabilization agent that causes the droplet to destabilize andto coalesce. Coalescence of the droplets can result in the generation ofa pooled mixture (e.g., a common pool) comprising the contents of thedroplets, including target molecules and any other contents of thedroplets (e.g., non-target molecules such as enzymes, additionalreaction products, reaction by-products reactants, co-factors, buffers,etc.). In general, the pooled mixture is a liquid mixture. Where thedroplets are aqueous droplets, the pooled mixture may comprise anaqueous mixture. The pooled mixture may also comprise any amount ofcontinuous phase (e.g., oil) material in which the droplets wereoriginally dispersed surfactants in the continuous phase, and/or thedestabilization agent where applicable. For example, in the case of anaqueous droplet or aqueous droplets comprising a degraded polyacrylamidebead and target molecules and originally dispersed in a fluorinated oil,the pooled mixture may comprise one or more of linear polyacrylamidefrom the degraded beads, the target molecules and the fluorinated oil.

In some examples, a droplet or plurality of droplets may be collectedinto a vessel and the destabilization agent may be added to the vesselto form the pooled mixture in the vessel. The term “vessel” as usedherein means any container that can hold a liquid mixture. A vessel mayinclude, without limitation, a droplet, a tube, a well, a container, adish, a flask, a beaker, and the like.

The destabilization agent can be any agent that induces droplets of theemulsion to coalesce with one another. The destabilization agent may bepresent at an amount effective to induce coalescence, which may beselected based, for example, on the volume of the emulsion, the volumeof carrier fluid in the emulsion, and/or the total volume of droplets,among others. The amount also or alternatively may be selected, based,for example, on the type of carrier fluid, amount and type of surfactantin each phase, etc. In exemplary embodiments, the destabilization agentcan be added to an emulsion, or vice versa, such that thedestabilization agent is present in excess over the continuous phase ofthe emulsion. The ratio of destabilization agent to continuous phase, byvolume, may be at least about 1, 2, 3, 4, or 5, among others. In someembodiments, the destabilization agent may be a fluid.

The destabilization agent may be a weak surfactant. Without wishing tobe bound by theory, a weak surfactant can compete with dropletsurfactant at the oil/aqueous interface causing an emulsion to collapse.In some cases, the destabilization agent is perfluorooctanol (PFO),however, other fluorous compounds with a small hydrophilic group may beused. Other examples of destabilization agents include one or morehalogen-substituted hydrocarbons. In some cases, the destabilizationagent may be predominantly or at least substantially composed of one ormore halogen-substituted hydrocarbons. Each halogen-substitutedhydrocarbon may be substituted with one or more halogen substituentsprovided by the same halogen element (i.e., one or more fluorine,chlorine, bromine, iodine, or astatine substituents) and/or two or moredifferent halogen elements (e.g., at least one fluorine substituent andat least one chlorine substituent, at least one fluorine substituent andat least one bromine substituent, at least one chlorine substituent andat least one bromine substituent, and so on). The halogen-substitutedhydrocarbon also optionally may include other non-halogen substituents.In some cases, the halogen-substituted hydrocarbon may have a formulaweight of less than about 1000, 500, or 200 daltons, among others. Alsoor alternatively, the halogen-substituted hydrocarbon may be composed ofno more than ten, five, or two carbons. Exemplary halogen-substitutedhydrocarbons that may be included in the destabilization agent includechloroform, dichloromethane (methylene chloride), iodomethane,bromochloropropane, or dichlorofluoroethane, among others. Thedestabilization agent may have a low viscosity and may be capable ofdenaturing proteins present in the droplets and/or at an interfacebetween the droplets and the carrier fluid. Additional examples ofdestabilization agents are provided in U.S. Pat. Publication No.2013-018970, the full disclosure of which is incorporated herein byreference for all purposes.

Purifying Target Molecules From a Pooled Mixture

A target molecule(s) partitioned into a droplet(s) may be recovered foruse in downstream applications (e.g., target nucleic acid molecules forsequencing). However, the success of downstream applications can beaffected by one or more non-target molecules present in the droplets oremulsion comprising the droplets and, thus, a pooled mixture asdescribed above. In some cases, the presence of one or more contaminantsin a pooled mixture can negatively impact the success of downstreamapplications. Therefore, it may be desirable to purify the targetmolecule(s) from one or more non-target molecule(s) or “contaminants”. A“contaminant” as used herein can generally refer to any non-targetsubstance (i.e., chemical, biological, or otherwise) derived from thecontents of a droplet or droplets and present in a pooled mixturegenerated as described elsewhere herein. Contaminants can include,without limitation, one or more oils (e.g., fluorinated oils), salts,biological materials (e.g., nucleic acids, proteins, lipids, etc),surfactants, polymers (e.g., polymers from degraded polymeric particles,such as linear polyacrylamide from a degraded polymeric particle),reactants, destabilization agents, reaction by-products, additionalreaction products, enzymes (e.g., polymerases), primers, co-factors andthe like. Contaminants can be components of the droplets, components ofthe partitioning fluids, components of an enzymatic reaction (e.g., PCRreagents, for example, enzymes, primers, dNTPs, and the like), etc.

Target molecules may be purified from contaminants in a pooled mixtureby contacting the target molecules with one or more supports andisolating the supports from the pooled mixture. For example, the one ormore supports can be provided to the pooled mixture or a vessel (asdescribed elsewhere herein) comprising the pooled mixture. In suchcases, the pooled mixture may comprise the contents of one or moredroplets and one or more supports. A support generally refers to anyspecies (e.g., a scaffolding or platform) that can selectively bind oneor more target molecules in a pooled mixture. In some cases, the supportmay be solid or, in other cases, the support may be liquid. Moreover,the support may be essentially of any shape, size or material. In somecases, a support may be a particle, such as, for example, a bead.

The support may have one or more surfaces on which target molecules canbind and is generally separable from the pooled mixture. For example,the support may bind a target molecule in a pooled mixture and then thesupport can be isolated or separated from the non-target molecules suchthat the target molecule is also separated from the pooled mixture. Atarget molecule that has been bound to a support can be referred toherein as a “bound target molecule”.

Binding of target molecules to a support may be through any suitablemeans, including ionic interactions, hydrophilic/hydrophobicinteractions, van der Waals forces, covalent bonds, non-covalent bonds,etc. Binding of a target molecule to the surface of a support may bespecific or non-specific. Binding of a target molecule to a support maybe specific for a class of target molecules (e.g., specific for nucleicacid molecules such as DNA). In this example, all or substantially allnucleic acid may be bound to the support. In other examples, the supportmay bind to a specific species of target molecule. In yet furtherexamples, the support may selectively bind to target molecules of aparticular size (e.g., the support may selectively bind to nucleic acidsgreater than 100 basepairs). The surface of the support may inherentlybind to a class of target molecules or the surface of the support may bemodified to bind to a class of target molecules.

In some examples, a support may be tailored such that particular targetmolecules can bind. For example, in the case where a target molecule isa nucleic acid (e.g., DNA, mRNA, etc) the surface of a support cantailored to bind nucleic acids by functionalizing the support with oneor more species capable of binding nucleic acids. A variety of methodsfor modifying the surface of a support such that said surface can bindnucleic acids are known. For instance, the support may be modified byany number of polycations. The polycationic support can be selected froma wide variety of inorganic and organic materials including, but notlimited to, metal oxides, glasses, polyamides, polyesters, polyolefins,polysaccharides, polyglycols, and polyaminoacids. In some cases, thesupport may be a silica bead or silica resin. In some cases, the bindingof target nucleic acid molecules to one or more supports may be via oneor more ionic interactions.

Supports may be modified with any number of polymers that bind nucleicacids. By way of example, without limitation, polymers that bind nucleicacids include dextran, polyethylene glycol (PEG), polyvinylpyrrolidone(PVP), polysaccharides (e.g., include dextran, ficoll, glycogen, gumarabic, xanthan gum, carageenan, amylose, agar, amylopectin, xylans,beta-glucans, and many others), chemical resins (isocyanate, glycerol,piperidino-methyl, polyDMAP (polymer-bound dimethyl 4-aminopyridine),DIPAM (Diisopropylaminomethyl), aminomethyl, polystyrene aldehyde,tris(2-aminomethyl) amine, morpholino-methyl, BOBA (3-Benzyloxybenzaldehyde), triphenyl-phosphine, and benzylthio-methyl, and others).It should be understood that surfaces modified with polymers orfunctional groups can, with some exceptions, bind target nucleic acidmolecules without regard to nucleic acid sequence. However, methods inwhich nucleic acids specifically bind to the surface of a support (i.e.,sequence-dependent binding) are also contemplated. In these examples, anoligonucleotide probe may be attached (i.e., via chemical modification)to a support. The oligonucleotide probe may contain a sequence ofnucleotides that can selectively bind to a target nucleic acid moleculesequence present in the pooled mixture.

Additional examples of functional groups with which a support may befunctionalized include, without limitation, alkanes, alkenes, alkynes,diene, alcohols, ethers, aldehydes, ketones, carboxylic acids, esters,amides, amines, nitriles, thiols, silanols, and the like. In someexamples, a support may comprise a silica or silica-like (i.e., modifiedto incorporate silanol on the surface) functionalization. In some cases,the support can bind nucleic acids. In some cases, the surface of thesupport can be silica-like and the support can bind nucleic acids.

In some instances, it may be desirable to bind other target molecules tothe surface of a support. For example, the target molecule may be asmall molecule, a protein, a peptide, and the like. In some aspects, thetarget molecule may be a protein. Methods of binding proteins to solidsupports are well known. For example, the surface of the support may becoated with antibodies and the antibodies may recognize a specificepitope of a protein. In another example, the support may be coated withstreptavidin and the target protein molecule may contain a biotinmolecule. Streptavidin has a high affinity for biotin and is a commonlyused method for pulling down proteins modified with biotin moieties.Other suitable examples may include coating the support with a proteinthat interacts with the target protein, for example, a surface coatedwith a receptor protein to bind a ligand present in the pooled mixture.

It may be desirable to provide suitable binding conditions to promotebinding of a target molecule to a support. A number of factors canaffect the conditions suitable for binding a target molecule to asupport. Non-limiting examples of such factors include the surfacemodification of the support, the type of target molecule, and thecomposition of the pooled mixture (i.e., chemicals, non-targetmolecules, etc., present in the pooled mixture). In some embodiments,the pooled mixture may comprise one or more additional agents that aidin binding target molecules to supports. Non-limiting examples of suchagents include buffer salts, detergents, enzymes, nuclease inhibitors,chelators, organic solvents, and other organic or inorganic substances.

In some examples, an agent that aids in binding target molecules to oneor more supports may comprise a chaotropic agent or chaotrope.“Chaotropic agent” and “chaotrope” are used interchangeably herein. Anysuitable concentration of chaotropic agent may be used. For example, theconcentration of chaotropic agent used may be from about 0.01 molar (M)to about 20 M. In some cases, the concentration of chaotropic agent usedmay be from about 0.1 M to about 10 M. In some cases, the concentrationof chaotropic agent used may be from about 1 M to about 8 M. In somecases, the concentration of chaotropic agent used may be from about 1 Mto about 5 M. In some cases, the concentration of chaotropic agent usedmay be about 0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 1.0 M,1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M, 4.5 M, 5.0 M, 5.5 M, 6.0 M,6.5 M, 7.0 M, 7.5 M, 8.0 M, 8.5 M, 9.0 M, 9.5 M, 10.0 M, 10.5 M, 11.0 M,11.5 M, 12.0 M, 12.5 M, 13.5 M, 14.0 M, 14.5 M, 15.0 M, 15.5 M, 16.0 M,16.5 M, 17.0 M, 17.5 M, 18.0 M, 18.5 M, 19.0 M, 19.5 M, 20.0 M or more.

In some cases, the chaotropic agent may comprise a chaotropic salt.Binding of the nucleic acids on a substrate in the presence ofchaotropic reagents may be that adsorption of nucleic acids to asubstrate lie in disturbances of higher-order structures of the aqueousmedium. Such disturbance can lead to adsorption or denaturation ofdissolved nucleic acid on the surface of the glass or silica-gelparticles. In the presence of chaotropic salts, such adsorption isenhanced. In some examples, binding of target molecules in a pooledmixture may be binding with a silica particle or silica-coated particlein the presence of a chaotropic salt. In such examples, the targetmolecule may be a nucleic acid. Moreover, in some cases, polyethyleneglycol (PEG) may be present in a pooled mixture to aid in the binding oftarget molecules (e.g., target nucleic acid molecules) to one or moresupports.

In some examples, the chaotropic agent is guanidine thiocyanate (GuSCN),however, essentially any chaotropic agent may be used. In otherexamples, the chaotropic agent is guanidine hydrochloride (GuHCl). Othernon-limiting examples of chaotropic agents include, without limitation,urea, sodium perchlorate, lithium perchlorate, lithium acetate, lithiumchloride, magnesium chloride, sodium acetate, potassium acetate,potassium chloride, sodium iodide, sodium chloride, ethanol, isopropanoland combinations thereof.

In some cases, a support may comprise at least one component, such asone or more magnetic materials, that is responsive to a magnetic force.In some cases, a support may be entirely magnetic. In some cases, asupport may be a magnetic particle, such as a magnetic bead. In suchcases, the magnetic particle may be entirely magnetic or may compriseone or more magnetic cores surrounded by one or more additionalmaterials, such as, for example, one or more functional groups and/ormodifications for binding one or more target molecules.

In some examples, a support may comprise a magnetic component and asurface modified with one or more silanol groups. Supports of this typemay be used for binding target nucleic acid molecules. Silanol-modifiedmagnetic beads are commercially available (AccuBead silica-coatedmagnetic beads available from Bioneer, silane-modified Dynabeadsavailable from Life Technologies, MagSi beads available from AMSBIO,among others). In some examples, a support may be a magnetic bead orparticle and the surface may be functionalized with a plurality ofcarboxyl groups. Such supports can make use of solid phase reverseimmobilization (SPRI) technology. Carboxylated magnetic beads areavailable from commercial sources, for example. Agencourt AMPure XP SPRIbeads available from Beckman-Coulter.

Magnetic materials may be classified according to their magneticproperties. Without wishing to be bound by theory, materials cangenerally be classified as diamagnetic, paramagnetic, or ferromagnetic.Diamagnetism is a property of all materials and can be a weak magneticforce. When diamagnetism is a magnetic property of a material, thematerial can be considered “diamagnetic.” Diamagnetic materials cancreate an induced magnetic field in a direction opposite to anexternally applied magnetic field. Paramagnetic materials can beattracted by an externally applied magnetic field and form inducedmagnetic fields in the direction of the applied magnetic field,Ferromagnetic materials are those that can be become permanentlymagnetized in the presence of a magnetic field. Examples of magneticmaterials that may be included in a support include iron, nickel,cobalt, composites thereof and alloys thereof. In some cases, a magneticmaterial may include one or more iron-oxides, such as magnetite ormaghemite.

One or more magnetic supports can be isolated and/or immobilized byapplying a magnetic force to the one or more supports (e.g., magneticseparation). A magnetic force can be applied to one or more supports byexposing the one or more supports to an external magnetic field. Such anexternal magnetic field may be provided by one or more magnetic sourcessuch as, for example, by one or more magnets (e.g., permanent magnetic,electromagnet, etc.). The magnetic responsiveness of a support to amagnetic force can be useful in isolating a support having bound targetmolecules from a pooled mixture. Application of a magnetic force to thesupport can result in separation of the support from other components ina pooled mixture. Accordingly, any molecule (e.g., one or more targetmolecules) that is also bound to the support, covalently ornon-covalently, can also be separated from non-bound components in apooled mixture. When an external magnetic field is applied to a support,the support can be attracted via magnetic force in the direction of theexternal magnetic field. The source of an external magnetic field, suchas one or more magnets, can be positioned such that a support or aplurality of supports is attracted to one or more specific locations.For example, when one or more supports are provided to a pooled mixturein a vessel, the one or more supports may be positioned at one or morelocations (e.g., surfaces) of the vessel. For example, a magnetic sourcecan immobilize a support at the bottom of a vessel. In some cases, amagnetic source can immobilize a support on a wall of a vessel.

Magnetic immobilization/separation of one or more supports at multiplepositions within a vessel may be used in purification. Magneticseparation at multiple positions within a vessel may occursimultaneously (e.g., one or more supports simultaneously positioned ata plurality of locations within a vessel) or sequentially (e.g., a firstround of magnetic separation at a first location, a second round ormagnetic separation at a second location, etc.). For example, one ormore magnetic supports may be provided to a vessel comprising a liquidpooled mixture comprising contents of one or more destabilized dropletsthat comprise one or more target molecules. The one or more supports canbind the target molecule(s) to provide a bound target molecule(s).Following binding of target nucleic acid molecules to the one or moresupports, the one or more supports can be immobilized at a firstlocation of the vessel via an external magnetic field as describedelsewhere herein, thereby separating or isolating the one or moresupports (and associated target nucleic acid molecules) from the pooledmixture. For example, an external magnetic field may be applied to thevessel such that a magnetic support comprising a bound target moleculewithin the vessel is attracted or pulled towards the first location. Thefirst location may be any portion of a wall of the vessel or the bottomof the vessel. Immobilized supports may be in the form of a pellet atthe bottom or on the wall of a vessel. Generally, an immobilized supportcan be segregated in a vessel and the movement of the immobilizedsupport can be restricted (e.g., to the bottom of or the wall of avessel).

Next, the remnant liquid pooled mixture can be removed from the vessel,without removing immobilized support(s) from the vessel. Removal of theliquid pooled mixture from the vessel may be by any method known ofremoving a liquid from a vessel, including, but not limited to,pipetting, suctioning, decanting, pouring, and the like. A suspensionfluid may then be provided to the vessel and the external magnetic fieldmay be removed from the supports, thereby suspending the support in thesuspension fluid. A suspension fluid may be any fluid, aqueous orotherwise, that can be used to release the support from an immobilizedstate. Essentially any fluid may be used as a suspension fluid, althoughgenerally, the suspension fluid can be selected such that it iscompatible with the support and the target molecule and such that itwill not disrupt the binding of the target molecule to the support. Insome cases, the suspension fluid may promote the binding of a targetmolecule to the support. In some examples, the target molecule may be atarget nucleic acid molecule and the suspension fluid may comprise achaotrope (e.g., guanidine thiocyanate or guanidine hydrochloride). Insome cases, the suspension fluid may be a washing agent (e.g., ethanol,isopropanol, acetone, etc.) as described elsewhere herein. Suspending asupport may involve the addition of a suspension fluid to the vessel andthen physically agitating the support, such as by pipetting orvortexing.

The suspended support(s) comprising the bound target molecule(s) maythen be immobilized at a second location of the vessel, therebyseparating the support(s) and associated bound target molecule(s) fromthe suspension fluid. The second location of the vessel may be the samelocation as the first location of the vessel or may be a different thanthe first location of the vessel. For example, an external magneticsource may be applied to the vessel such that the magnetic supportcomprising a bound target molecule within the vessel is attracted orpulled towards the second location. The second location may be anyportion of a wall of the vessel or may be at the bottom of the vessel.Immobilizing a magnetic support at a first location and a differentsecond location may be accomplished by adjusting the location of theapplied external magnetic field (e.g., adjusting the positioning of oneor more magnetic sources providing the applied external magnetic field).Devices described elsewhere herein may be useful in adjusting thepositioning of one or more magnetic sources (and associated externalmagnetic fields).

One or more magnetic sources may be provided in a device that canreceive one or more vessels such that the magnetic source(s) immobilizeone or more supports to one or more surfaces of the one or more vessels.The device may comprise one or more holders or receptacles for receivingone or more vessels, where each holder or receptacle is associated withone or more magnetic sources that provide an external magnetic field toeach respective vessel. In some cases, such a magnetic source maycomprise one or more magnets. In such cases, the one or more magneticsources may be positioned in such a way that the one or more magneticsupports are drawn towards a particular portion of the vessel (e.g., asurface of the vessel, a wall of the vessel, the bottom of the vessel,etc.). In some cases, a position of a magnetic source in a device may beadjustable such that the external magnetic field generated by themagnetic source can be adjusted to immobilize one or more supports at avariety of locations within a vessel. In some examples, one or moremagnetic sources may be included in a sliding rack also a part of adevice. In such a configuration, the position of the one or moremagnetic sources may be adjusted by moving the sliding rack (e.g.,moving the sliding rack up and down) to a desired position. A device mayalso include a latch or other component suitable for immobilizing asliding rack at a desired position. A device having an adjustablemagnetic source can be useful in cases where magnetic separation atdifferent locations of a vessel, as described elsewhere herein, isdesirable. In some cases, a device may comprise a plurality of magneticsources associated with a receptacle for receiving a vessel, where eachof the magnets is positioned at a different location with respect to thereceptacle. In some cases, repositioning of a magnetic source andassociated external magnetic field within a device may be achieved byrepositioning the entire device, such as flipping the device over,rotating the device, etc. In some cases, repositioning of a vesselwithin a device may, with respect to the vessel, result in repositioningof an applied external magnetic field exerted by a magnetic source ofthe device. As can be appreciated any suitable combination ofrepositioning a magnetic source, repositioning of a device comprising amagnetic source, and repositioning a vessel within a device may be usedto position supports at multiple locations of a vessel. Moreover, adevice may comprise a plurality of receptacles and accompanying magneticsources such that a plurality of vessels can be processed in parallel.Any suitable number of receptacles and accompanying magnetic sources maybe included.

An example device suitable for performing magnetic separations,including those described herein, is schematically depicted in variousviews in FIGS. 6A-6C. FIGS. 6A and 6B show front (FIG. 6A) and back(FIG. 6B) views of an example magnetic separation device 600. As shownin FIGS. 6A and 6B, magnetic separation device 600 comprises a body 601that comprises a series of eight receptacles 602 on its top side eachcapable of receiving a vessel 603. As shown in FIGS. 6A and 6B, a seriesof vessels 603 may be provided to the device in a single strip, suchthat each vessel in the strip is positioned to be received by anindividual receptacle 602. The bottom side of device 600 also comprisesa series of eight receptacles (not shown in FIGS. 6A or 6B) 606 thatcorrespond to receptacles 602 on the top side of magnetic separationdevice 600. Moreover, as shown in FIG. 6A, the front side of magneticseparation device 600 comprises a series of receptacles 604 that eachcorrespond to one of the receptacles 602. Each of these receptacles iscapable of receiving and positioning one or more magnets 605 (e.g., twomagnets positioned back-to-back for magnetic device 600). Moreover, asshown in FIG. 6B, magnetic separation device 600 comprises a viewingwindow 607 for each of the receptacles 602. Such viewing windows 607 canaid in observing a magnetic separation in a vessel placed in areceptacle 602.

FIG. 6C shows a side view of magnetic separation device 600, showing avessel 603 placed in a receptacle 602. As shown in FIG. 6C. when vessel603 is placed in receptacle 602, magnets 605 are positioned near or atthe bottom of vessel 603. Magnets 605 can exert a magnetic field andapply a magnetic force to magnetic material (e.g., one or more magneticsupports) in vessel 603 such that the magnetic material is immobilizedon a wall near or at the bottom of the vessel 603. Moreover, magneticseparation device 600 also comprises receptacles 606 at its bottom side,such that when the device 600 is flipped over (e.g., rotatedapproximately 180 degrees with respect to the view shown in FIG. 6C),receptacles 606 are then positioned to receive vessels 603. When avessel 603 is placed in a receptacle 606, magnets 605 are positioned ata higher, different location of vessel 603 than when vessel 603 isplaced into receptacle 602. Magnets 605 can exert a magnetic field andapply a magnetic force to magnetic material (e.g., one or more magneticsupports) in vessel 603 such that the magnetic material is immobilizedat the higher location of vessel 603.

FIG. 6D shows an example use of magnetic separation device 600. As shownin FIG. 6D (top panel), magnetic separation device 600 is positionedsuch that receptacles 606 receive vessels 603. In this orientation ofmagnetic separation device 600, magnets 605 are positioned at a “high”location of vessels 603. Magnets 605 can exert a magnetic force onmagnetic material (e.g., one or more magnetic supports) in vessels 603such that the magnetic material is immobilized at the “high” location ofvessels 603. Magnetic separation in the windows can be observed throughviewing windows 607.

The vessels 603 can then be removed from magnetic separation device 600such that the immobilized magnetic material in vessels 603 is released.Release may be further facilitated by addition of a wash fluid andagitation (e.g., via vortexing or pipetting). Magnetic separation device600 may be flipped over 609 (e.g., rotated approximately 180 degrees)such that receptacles 602 receive vessels 603, as shown in FIG. 6D(bottom panel). In this orientation of magnetic separation device 600,magnets 605 are positioned at a “low” location (e.g., a location lowerthan the “high” location of vessels 603, such as at or near the bottomof the vessels) of vessels 603. Magnets 605 can exert a magnetic forceon magnetic material (e.g., one or more magnetic supports) in vessels603 such that the magnetic material is immobilized at the “low” positionof vessels 603. Magnetic separation in the windows can be observedthrough viewing windows 607.

While only magnetic separation device 600 is shown as capable ofprocessing up to eight vessels in parallel, it can be appreciated thatmagnetic separation device 600 could include any suitable number ofreceptacles and accompanying magnets for parallel processing of vesselsof fewer or greater numbers of vessels.

Another example device suitable for performing magnetic separations,including those described herein, is schematically depicted in FIGS. 7 .As shown in FIG. 7A, magnetic separation device 700 comprises a seriesof eight receptacles 701 and a series of eight corresponding receptacles702 each series capable of receiving vessels 703. When magneticseparation device 700 is in position 700A, receptacles 701 are capableof receiving vessels 703. The vessels 703 are positioned such that theyare each in proximity to a set of magnets (e.g., two magnets positionedback-to-back are shown for each set as in FIG. 7C), that are positionedto exert a magnetic force at a “high” position of the vessels 703. Sucha force can immobilize magnetic material (e.g., one or more supports) invessels 703 at the “high” position of the vessels 703. As shown in FIG.7C, each magnet of a magnet set is housed in magnetic separation device700 in a recess 705. Additional views (top 710, side 720 and bottom 730)of magnetic separation device 700 (when in position 700A) are shown inFIG. 7B.

Upon flipping magnetic separation device 700 over 706 (e.g., rotatingmagnetic separation device 700 approximately 180 degrees), magneticseparation device is in position 700B, as shown in FIG. 7A. Whenmagnetic separation device 700 is in position 700B, receptacles 702 arecapable of receiving vessels 703. The vessels 703 are positioned totheir corresponding set of magnets now positioned to exert a magneticforce at a “low” position of the vessels 703 (e.g., near or at thebottom of vessels 703). Such a force can immobilize magnetic material(e.g., one or more supports) in vessels 703 at the “low” position of thevessels 703.

Another example device suitable for performing magnetic separations,including those described herein, is schematically depicted in FIGS. 8 .As shown in FIG. 8A, magnetic separation device 800 comprises a body 801and a series of eight corresponding receptacles 802 each series capableof receiving vessels 803. Magnets 805 are included within receptacles804 as shown in FIG. 8A. Magnetic separation device 800 includes thefeatures and uses as described for the devices of FIGS. 6 and 7 . Inaddition, the magnetic separation device 800 includes scoops 806 tofacilitate access and physical manipulation of vessels 803 in use. Asshown in FIGS. 8A and 8B, concave scoops 806 can be position at distalends of body 801 and adjacent to receptacles 803.

Another example device suitable for performing magnetic separations,including those described herein, is schematically depicted in FIGS. 9 .As shown in FIG. 9A, magnetic separation device 900 comprises twoparallel series of eight receptacles 902, for a total of sixteenreceptacles each capable of receiving up to sixteen vessels 903,Magnetic separation device 900 includes the features and uses asdescribed for the devices of FIGS. 6, 7 and 8 . FIG. 9A additionallyshows interlocking ends 908 features for coupling multiple magneticseparation devices 900 in series.

FIG. 9B is a detail schematic of magnetic separation device 900, showingthe positioning of receptacles 902 and 906, in relation to vessels 903and magnets 905 within base 901.

FIG. 9C shows how interlocking ends 908 features can be connected forcoupling multiple magnetic separation devices 900 in series.

It should be understood that other methods of separating supports from apooled mixture can be used. In some examples, one or more supports maybe separated using centrifugation. In this case, one or more supportswith bound target molecules can be centrifuged such that the supportsare pelleted at the bottom of a vessel. Examples of support materialsuseful for centrifugation separation may include, without limitation,agarose beads, gel beads, glass beads, and the like. Essentially anysupport with a density greater than the mixture it is contained withincan be separated using centrifugation.

After one more supports comprising one or more bound target moleculesare separated from a pooled mixture, the non-separated components of thepooled mixture can be removed by, for example, removing the remnantmixture. Any method of removing the non-separated components of thepooled mixture may be used. Such methods of removing may include,without limitation, pipetting, decanting, pouring, suctioning (e.g., bya vacuum), evaporating, sublimating, vaporizing, and the like.

A support that is been separated from a pooled mixture may be washed inone or more cycles to further remove any remaining undesired componentsassociated with the supports. For example, prior to releasing a boundtarget molecule(s) from one or more supports, the supports may be washedin one or more wash steps by contacting the supports with a washingagent. In general, the supports can be washed with a washing agent thatis compatible with both the support and the bound target molecule. Thewashing agent can be selected such that it does not disrupt the bindingof the target molecule(s) to the support(s). For example, washing targetnucleic acids bound to a support may be carried out with an alcohol, forexample, ethanol or isopropanol. Alternatively, the washing agent may bean organic solvent (e.g., acetone). A washing step may comprise a stepof mixing the support with the washing agent (i.e., pipetting thesupport up and down, vortexing, etc.). In this case, another step ofseparating may be necessary to separate the support from the washingagent (e.g., reapplying a magnetic field to a vessel to separatemagnetic supports from the washing agent, centrifugation, etc.). Anysuitable separation method can be utilized including purificationmethods described herein. In some examples, the supports may not bewashed. In some instances, more than one wash step may be desired, e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, or more wash steps.

Following purification, bound target molecules can be recovered from theone or more supports for downstream applications or for furtherprocessing. Recovery can be achieved by releasing or eluting the boundtarget molecules from the one or more supports. For example, releasingbound target molecules may be may include containing the one or moresupports with an elution agent that aids in releasing bound targetmolecules from the supports. Contacting the one or more supports withelution agent may comprise mixing the supports with the elution agent(e.g., pipetting up and down, vortexing, etc.). An elution agent can beselected such that it can effectively release the bound target moleculesfrom the supports. Non-limiting examples of elution agents includewater. Tris buffer, phosphate buffer, and sodium hydroxide. In somecases, bound nucleic acid molecules can be eluted in a buffer of lowionic strength (e.g., TE buffer, or a similar buffer).

In some examples, release of target molecules from one or more supportsmay be achieved with heating of the supports. The supports may be heatedto 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58°C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67°C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76°C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85°C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94°C., 95° C., 96° C., 97° C., 98° C., 99° C., or 100° C. In some examples,releasing target molecules from supports may comprise adding an elutionagent to the supports and then heating the supports.

Target molecules released from supports may be further purified. Forexample, released target molecules (e.g., target nucleic acid molecules)and from the supports may be subjected to a solid phase reverseimmobilization (SPRI) process. For example, released target moleculesmay be contacted with SPRI beads in a mixture under appropriate bindingconditions such that the released target molecules bind to the SPRIbeads. In a matter similar to that described elsewhere herein, the SPRIbeads containing the bound target molecules may be subjected to anexternal magnetic field such that the SPRI beads are pulled towards theexternal magnetic field, thereby separating the SPRI beads from othercomponents of the mixture. The SPRI beads may optionally undergo one ormore washing steps as is discussed above. The target molecules can thenbe released from the SPRI beads via an elution agent and/or heating, asis discussed above.

It can be understood that further purification of one or more targetmolecules may be optional and can generally be based upon the level ofpurity desired for downstream applications. In some cases, a singleround of purification may be sufficient. For example, a method maycomprise binding target molecules to a single plurality of supports(e.g., magnetic particles such as Dynabeads), followed byseparation/isolation and any washing of the supports. In some cases, itmay be useful to combine a first round of purification and a secondround of purification. For example, a method may comprise binding targetmolecules to a first plurality of supports (e.g., magnetic particlessuch as Dynabeads), followed by separation/isolation and any washing ofthe first supports; releasing the target molecules from the firstsupports; and subsequently binding the target molecules to a secondplurality of supports (e.g., magnetic particles such as SPRI beads),followed by separation/isolation and any washing of the second supports.Additional rounds of purification need not be a SPRI process but maycomprise any other steps that have been disclosed herein. Moreover,greater than two rounds of purification may be completed, including upto 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20rounds of purification or more.

As can be appreciated, the purification methods described herein may beused to purify one or more target molecules from any suitable type ofpartition or plurality of partitions, including non-droplet partitions.A suitable partition can be any partition from which the contents of thepartition can be released and/o recovered. Non-limiting examples of suchpartitions include wells (e.g., microwells), microcapsules, tubes,containers, spots, microreactors, micelles and polymeric capsules.Moreover, the contents of a partition or a plurality of partitions maybe released from the partitions and pooled into a pooled mixture. One ormore target molecules may be recovered from the pooled mixture using oneor more purification methods as described herein.

Further Processing and Analysis of Purified Target Molecules

Target molecules that have been recovered from droplets and purified cansubject to further processing and/or analysis. In some cases,purification of target molecules can aid in performing cleaner or moreefficient further processing of target molecules. For example, apurified target nucleic acid molecule may function as a template for anamplification reaction, such as polymerase chain reaction or other typeof amplification reaction. One or more amplification reactions may becompleted using the purified target nucleic acid molecules in order toprovide an amplified number of target nucleic acid molecules. Suchfurther processing may be particularly useful where recovered, purifiednucleic acid molecules are initially present in low amounts and greatercopy numbers are needed for downstream analysis. Moreover, one or moreamplification reactions of purified target nucleic acid molecules may becompleted in bulk and may be used to add one or more additionalsequences (e.g., append additional nucleotides) to the purified targetnucleic acid molecules. Such additional sequences can result in thegeneration of larger nucleic acid molecules (e.g., larger target nucleicacid molecules) and the one or more added sequences may be one or morefunctional sequences. Non-limiting examples of such functional sequencesinclude a tag, a barcode sequence, an adapter sequence for sequencecompatibility with a sequencing instrument/protocol (e.g., P5, P7Illumina adaptor sequences), a primer (e.g., a random N-mer), asequencing primer binding site, a sample index sequence, etc. Examplesof adding additional sequences to nucleic acid molecules via anamplification reaction (including a bulk amplification reactions) areprovided in U.S. Pat. Application No. 14/316,383, filed Jun. 26, 2014and U.S. Provisional Pat. Application No. 62/102,420, filed Jan. 12,2015, the full disclosure of which is incorporated herein by referencein its entirety for all purposes.

In some cases, one or more additional sequences may be added to targetnucleic acid molecules (or amplified target nucleic acid molecules) viaa ligation process to generate larger target nucleic acid molecules. Insome cases, the target nucleic acid molecules may be subject to ashearing process in order to generate one or more ends of the targetnucleic acid molecules that are suitable for ligation with an additionalnucleic acid sequence. The additional nucleic acid sequence may compriseone or more of any of the functional sequences described herein.Examples of shearing and ligation methods that can be used for addingadditional sequences to nucleic acid molecules are provided in detail inU.S. Provisional Pat. Application No. 62/102,420, filed Jan. 12, 2015,the full disclosure of which is incorporated by reference in itsentirety for all purposes. Upon addition of the additional sequence(s)to the target nucleic acid molecules, the larger sequences that aregenerated can be amplified to provide greater copy numbers. Shearing,ligation and any subsequent amplification can be performed in bulk.

Purified target nucleic acid molecules (that may or may not be furtherprocessed) or purified nucleic acid molecules to which one or moreadditional sequences have been appended (e.g., larger target nucleicacid molecules) may be subject to nucleic acid sequencing, whereby asequence of the purified target nucleic acid molecules or larger targetnucleic acid molecules is determined. The addition of additionalfunctional sequences to purified target nucleic acid molecules may beuseful in preparing target nucleic acid molecules for sequencing.Purified target nucleic acid molecules may be prepared for any suitablesequencing platform and sequenced, with appropriate functional sequencesadded to purified target nucleic acid molecules where needed. Sequencingmay be performed via any suitable type of sequencing platform, withnon-limiting examples that include Illumina. Ion Torrent, PacificBiosciences SMRT. Roche 454 sequencing. SOLiD sequencing, etc. As can beappreciated, sequences obtained from nucleic acid molecules can beassembled into larger sequences from which the sequence of the nucleicacid molecules originated. In general, sequencing platforms make use ofone or more algorithms to interpret sequencing data and reconstructlarger sequences.

Methods described herein can be used to prepare and sequence a nucleicacid molecule library. In some cases, a library of nucleic acidmolecules can be generated, wherein the library comprises a plurality ofdroplets or other type of partitions comprising the nucleic acidmolecules. Examples of preparing a library of nucleic acid molecules inpartitions are provided in detail in e.g., U.S. Pat. Application No.14/316,383, filed June 26. 2014, U.S. Provisional Pat. Application No.62/017,808, filed Jun. 26, 2014 and U.S. Provisional Patent Application62/102,420, filed Jan. 12, 2015 (the full disclosures of which areincorporated by reference in their entireties for all purposes). Wherethe library of nucleic acid molecules comprises a plurality of dropletshaving the nucleic acid molecules, the plurality of droplets can bedestabilized, thereby releasing the nucleic acid molecules from theplurality of droplets into a common pool. The nucleic acid molecules(e.g., target nucleic acid molecules) can be recovered/purified from thecommon pool using one or more of any of the purification methodsdescribed herein. The purified nucleic acid molecules can optionally besubject to further processing as described elsewhere herein and subjectto sequencing, whereby the sequences of at least a subset of thepurified nucleic acid molecules (or further processed purified nucleicacid molecules) can be determined. Sequencing may be performed via anysuitable type of sequencing platform including example platformsdescribed elsewhere herein.

Kits

The disclosure further provides for one or more kits. The one or morekits may comprise the reagents and/or devices sufficient for performingthe methods provided in this disclosure. For example, the one or morekits may comprise the reagents and/or devices sufficient for purifying atarget nucleic acid molecule from a droplet or other type of partition.Accordingly, the one or more kits may include one or more of thefollowing reagents, without limitation: a destabilization agent, achaotrope, a washing agent, an elution agent, and a support (e.g., amagnetic support). In some instances, the one or more kits may comprisea device (e.g., a magnetic device) for separating supports from a pooledmixture. In some cases, a kit may comprise reagents suitable forgenerating an emulsion. Non-limiting examples of such reagents include acontinuous phase (e.g., oil) and an aqueous phase (e.g., a buffer). Theone or more kits may further comprise packaging (i.e., a box). Thereagents and the device may be packaged into a single kit.Alternatively, the reagents and the device may be packaged separately.The kits may further comprise instructions for usage of the kit. Theseinstructions may be in the form of a paper document or booklet containedwithin the packaging of the kit. Alternatively, the instructions may beprovided electronically (i.e., on the Internet).

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Example Workflow

FIG. 2 illustrates an example workflow for generating and sequencing anucleic acid library. As shown in FIG. 2 , the example workflow providesfor obtaining sample nucleic acids, generating barcoded nucleic acidsfrom the sample nucleic acids, purifying the barcoded nucleic acids, andsubsequently sequencing the barcoded nucleic acids. First, a samplecomprising nucleic acids may be obtained from a source, 200, and a setof barcoded beads may also be obtained, 210. The beads can be linked tooligonucleotides containing one or more barcode sequences, as well as aprimer, such as a random N-mer or other primer. The barcode sequencescan be releasable from the barcoded beads, e.g., through cleavage of alinkage between the barcode and the bead or through degradation of theunderlying bead to release the barcode, or a combination of the two. Forexample, in some cases, the barcoded beads can be degraded or dissolvedby an agent, such as a reducing agent to release the barcode sequences.In this example, sample comprising nucleic acids, 205, barcoded beads,215. and optionally other reagents, e.g., a reducing agent, 220, arecombined and subjected to partitioning. By way of example, suchpartitioning may involve introducing the components to a dropletgeneration system, such as a microfluidic device, 225. With the aid ofthe microfluidic device 225, a water-in-oil emulsion 230 may be formed,wherein the emulsion contains aqueous droplets that contain samplenucleic acid, 205, reducing agent, 220, and barcoded beads, 215. Thereducing agent may dissolve or degrade the barcoded beads, therebyreleasing the oligonucleotides with the barcodes and random N-mers fromthe beads within the droplets, 235. The random N-mers may then primedifferent regions of the sample nucleic acid, resulting in amplifiedcopies of the sample after amplification (e.g., target nucleic acidmolecules), wherein each copy is tagged with a barcode sequence. 240(“barcoded nucleic acids”). In some cases, each droplet may contain aset of oligonucleotides that contain identical barcode sequences anddifferent random N-mer sequences. Subsequently, the emulsion is brokento form a pooled mixture, 245 in a vessel. A plurality of magneticsupports is contacted with the pooled mixture in the presence of achaotrope such that the barcoded nucleic acids bind to the magneticsupports, 250. An external magnetic field is applied to the pooledmixture such that the magnetic supports are pelleted, 255. Thesupernatant is removed and the barcoded nucleic acids are released fromthe magnetic supports, 260 via the action of one or more elution agents.The process of applying an external magnetic field and pelleting thesupports may be repeated for one or more cycles (with the addition andremoval of fluid in each cycle) prior to release of the barcoded nucleicacids from the beads. The released barcoded nucleic acids may be subjectto further processing. 265. For example, additional sequences (e.g.,sequences that aid in particular sequencing methods, additionalbarcodes, etc.) may be added to the barcoded nucleic acids, via, forexample, amplification methods (e.g., PCR or other amplificationreaction) and/or ligation methods. Sequencing may then be performed onthe barcoded nucleic acids, 270. and one or more algorithms applied tointerpret the sequencing data, 275. Sequencing algorithms can be, forexample, of performing analysis of barcodes to align sequencing readsand/or identify the sample from which a particular sequence readbelongs.

Example 2: Example Purification Methods

FIGS. 3A-3F provide an example method for purifying target nucleic acidmolecules from aqueous droplets in an emulsion using magnetic supportsand a magnetic separation device. Aqueous droplets encapsulating aplurality of target nucleic acid molecules in an emulsion were collectedinto a high profile strip comprising 8 tubes. Each tube in the stripcontained an emulsion comprising a plurality of aqeuous droplets influorocarbon oil (having a fluorosurfactant) comprising a plurality oftarget nucleic acid molecules. The droplets had an approximate volume of360 picoliters (pL). 125 microliters (µL) of perfluorooctanol (PFO) wasadded to each tube and the strip was vortexed to to destabilize theemulsion and the droplets (FIG. 3A), resulting in the release of thecontents of the droplets into a pooled mixture in each tube. The oil/PFOphase separated to the bottom of the tubes/pooled mixtures and theaqueous phase containing the target nucleic acid molecules separated tothe top of the tubes/pooled mixtures. 135 µL of the oil/PFO phases wascarefully pipetted out of each tube so as not to disrupt the aqueousphase (FIG. 3B). As shown in FIG. 3B, each tube contained a smallremnant volume of oil/PFO at its bottom after removal of the 135 µL ofthe oil/PFO phases.

180-200 µL of a mixture comprising 3.5 µg/µL silanized Dynabeads (LifeTechnologies) and a chaotropic salt (5.5 M), guanidine thiocyanate(GuSCN) were added to each tube and the mixtures were mixed via pipetingto bind target nucleic acid molecules to the Dynabeads (FIG. 3C). Afteran incubation period of 10 min, the strip of tubes was placed into amagnetic separation device (FIG. 3D). As shown in FIGS. 3D-F, themagnetic separation device comprised a plurality of receptacles eachassociated with a set of magnets (e.g., two magnets positionedcontacting each other back-to-back). Each receptacle was capable ofreceiving a tube of the strip, such that the strip of tubes was placedin the device and each respective magnet set exerted a magnetic force onDynabeads in respective tubes. The positioning of the device as a wholedetermined whether each respective magnet set was in a “high” or “low”position with respect to each receptacle/tube. In a first position ofthe device, the magnet sets were positioned at a “high” position. Aftera 4 minute incubation in the magnetic device, the Dynabeads were pulledto the walls of the tubes (high position) such that the Dynabeads wereisolated from the pooled mixture. The pooled mixture (e.g.,supernatant)was pipetted out of the tube leaving behind a pellet of Dynabeadsattached to each of the walls of the tubes.

The Dynabeads were washed with ethanol (EtOH) amd the tubes removed fromthe device resulting in resuspension of the beads in the EtOH in eachtube. After the EtOH wash, the magnetic device was flipped over and thestrip of tubes was placed back into the device. At this second positionof the device, the magnet sets were positioned at a “low” position.After another 4 minute incubation, the Dynabeads were pulled to bottomwalls of the tubes (low position) (FIG. 3E). The EtOH was removed andthe Dynabeads were again washed with EtOH. The strip of tubes was onceagain placed into the second side of the magnetic device such that theDynabeads were pulled to the bottom of the strip tube (low position)(FIG. 3F). The EtOH wash was removed. The target nucleic acid moleculeswere eluted off of the Dynabeads with 52 µL of elution buffer comprising10 mM Tris-HCl buffer at pH 8.5 (FIG. 3F).

The target nucleic acid molecules were optionally further processed withSPRI beads. In this optional step, the target nucleic acid moleculeswere contacted with SPRI beads in the presence of polyethylene glycol(PEG) to bind the target nucleic acid molecules to the SPRI beads. TheSPRI beads underwent another round of magnetic separation and washingsteps as described above. The target nucleic acid molecules were elutedfrom the SPRI beads and collected, subjected to further processing toadd additional sequences suitable for Illumina sequencing and sequencedon an Illumina HiSeq 2500 sequencer.

Example 3: Example Purification Methods

To test example one-step purification methods (e.g., purification withonly one set of supports) versus an example two-step purification method(e.g., purification with a first set of supports, followed by furtherpurification with a second set of supports), the example methods werecompared side-by-side. 2 ng of 20kb DNA was co-partitioned with barcodedpolyacrylamide beads (e.g., beads comprising primers having a barcodesequence and a random N-mer primer sequence as described elsewhereherein) in aqueous droplets within a fluorinated oil continuous phasehaving a fluorosurfactant using a microfluidic partitioning system (See,e.g., U.S. Pat. Application No. 61/977,804, filed Apr. 4, 2014, andincorporated herein by reference in its entirety for all purposes),where the aqueous droplets also included dNTPs, thermostable DNApolymerase and other reagents for carrying out amplification within thedroplets, as well as a chemical activator (e.g., reducing agent) forreleasing the barcode oligonucleotides from the beads.

Following bead dissolution (e.g., via disruption of disulfide bonds ofthe beads), the droplets were thermocycled to allow for primer extensionof the barcode oligonucleotides against the template of the samplenucleic acids within each droplet. This resulted in the generation ofbarcoded copy fragments of the sample nucleic acids that included thebarcode sequence representative of the originating partition, asdescribed elsewhere herein. Four replicate samples of droplets havingbarcoded copy fragments were generated.

After generation of the barcoded copy fragments, the emulsion in eachsample was destabilized with the addition of perfluorooctanol (PFO).This resulted in destabilization of the droplets and the generation ofpooled mixture containing the contents of the droplets in each sample.Purification of the barcoded copy fragments from the pooled mixtures wasperformed using a magnetic device as described in Example 2 above andusing 4 different purification methods, similar to those described inExample 2. Three of the methods (Method 1, Method 2, Method 3) includedbinding of barcoded copy fragments to varied amounts of single set ofDynabeads in a single purification step, whereas the fourth methodincluded a two-step purification, whereby barcoded copy fragments werebound to a first set of Dynabeads, released and then bound to a secondset of SPRI beads. Each of the four samples was processed with one ofthe four purification methods, where the amount of Dynabeads added wascontrolled by volume of Dynabead and chaotrope mixture (as in Example 2)added to pooled mixtures. A summary of the methods is as follows:

-   Method 1: One-step: Dynabeads only (200µL mixture added) (D 200µL in    FIG. 4A)-   Method 2: One-step: Dynabeads only (190µL mixture added) (D 190µL in    FIG. 4A)-   Method 3: One-step: Dynabeads only (180µL mixture added) (D 180µL in    FIG. 4A)-   Method 4: Two-step: Dynabeads (200µL mixture added) followed by SPRI    beads (D 200µL + SPRI in FIG. 4A)

Purified barcoded copy fragments were analyzed for yield and thoseobtained from Methods 1 and 4 were subject to further processing to addappropriate adaptors to the purified barcoded copy fragments forIllumina Sequencing followed by whole genome sequencing (WGS) of thenucleic acid molecules on an Illumina sequencer. Data obtained from theexperiments in shown in FIGS. 4 . FIG. 4A graphically depicts productyields of the various purification methods. Methods 1 and 2 generatedhigher product yields than Methods 3 and 4, suggesting that a one-steppurification method could result in higher yields when compared to atwo-step purification method or other one-step purification methodsemploying a lower amount of Dynabeads. Moreover, Methods 1-4 wereeffective in lowering linear polyacrylamide (LPA) (e.g., resulting fromdissolution of polyacrylamide beads) from the barcoded copy fragments,which aided in further processing of purified copy fragments to addappropriate adaptors for sequencing.

FIG. 4B graphically depicts sequencing data obtained from theexperiments and further suggests that a one-step purification method canbe comparable to or even an improvement over a two-step method. As shownin FIG. 4B, the table illustrates the results of sequencing on thebarcoded, copy amplified fragments of Method 1 and Method 4. Method1 hada higher effective amplification rate and a higher effective barcodediversity as compared to Method 4 and similar unmapped fractions duringsequencing.

Example 4: Example Purification Methods

To test an example one-step purification methods (e.g., purificationwith only one set of supports) versus an example two-step purificationmethod (e.g., purification with a first set of supports, followed byfurther purification with a second set of supports), the example methodswere compared side-by-side. 1 ng of 20kb DNA was co-partitioned withbarcoded polyacrylamide beads (e.g., beads comprising primers having abarcode sequence and a random N-mer primer sequence as describedelsewhere herein) in aqueous droplets within a fluorinated oilcontinuous phase having a fluorosurfactant using a microfluidicpartitioning system (See, e.g., U.S. Pat. Application No. 61/977,804,filed Apr. 4, 2014, and incorporated herein by reference in its entiretyfor all purposes), where the aqueous droplets also included dNTPs,thermostable DNA polymerase and other reagents for carrying outamplification within the droplets, as well as a chemical activator(e.g., reducing agent) for releasing the barcode oligonucleotides fromthe beads.

Following bead dissolution (e.g., via disruption of disulfide bonds ofthe beads), the droplets were thermocycled to allow for primer extensionof the barcode oligonucleotides against the template of the samplenucleic acids within each droplet. This resulted in the generation ofbarcoded copy fragments of the sample nucleic acids that included thebarcode sequence representative of the originating partition, asdescribed elsewhere herein. Six replicate samples of droplets havingbarcoded copy fragments were generated.

After generation of the barcoded copy fragments, the emulsion in eachsample was destabilized with the addition of perfluorooctanol (PFO).This resulted in destabilization of the droplets and the generation of apooled mixture containing the contents of the droplets for each sample.Purification of the barcoded copy fragments from the pooled mixtures wasperformed using a magnetic device as described in Example 2 above andusing 3 different purification methods, similar to those described inExample 2. Two purification methods included binding of barcoded copyfragments to varied volumes of a single set of Dynabeads in a singlepurification step, whereas the third method included a two-steppurification, whereby barcoded copy fragments were bound to a first setof Dynabeads, released and then bound to a second set of SPRI beads.Each of the six samples was processed with one of the three purificationmethods, where the amount of Dynabeads added was controlled by volume ofDynabead and chaotrope mixture (as in Example 2) added to pooledmixtures. A summary of the methods is as follows:

-   Method 1: One-step: Dynabeads only (200 µL mixture added)-   Method 2: One-step: Dynabeads only (190 µL mixture added)-   Method 3: Two-step: Dynabeads (200 µL mixture added) followed by    SPRI beads

Purified barcoded copy fragments were analyzed for yield. Data obtainedfrom the experiments in shown in FIG. 5 . FIG. 5 graphically depictsproduct yields of the various purification methods. Both one-stepmethods (Method 1 and 2) generated higher yields than the two-stepmethod (Method 3). The data suggest that a one-step purification methodcould result in higher yields when compared to a two-step purificationmethod.

Example 5: Example Purification Methods

Genomic DNA from the NA12878 human cell line is subjected to size basedseparation of fragments using a Blue Pippin DNA sizing system to recoverfragments that are approximately 10kb in length. The size selectedsample nucleic acids are then copartitioned with barcoded beads inaqueous droplets within a fluorinated oil continuous phase using amicrofluidic partitioning system (See, e.g., U.S. Pat. Application No.61/977,804, filed Apr. 4, 2014, the full disclosure of which isincorporated herein by reference in its entirety for all purposes),where the aqueous droplets also include dNTPs, thermostable DNApolymerase and other reagents for carrying out amplification within thedroplets, as well as a chemical activator (e.g.. a reducing agent) forreleasing the barcode oligonucleotides from the beads. This is repeatedboth for 1 ng of total input DNA and 2 ng of total input DNA. Thebarcoded beads are obtained as a subset of a stock library thatrepresents barcode diversity of over 700,000 different barcodesequences. The barcode containing oligonucleotides include additionalsequence components and have the general structure:

Bead-P5-BC-R1-Nmer

Where P5 and R1 refer to the Illumina attachment and Read1 primersequences, respectively, BC denotes the barcode portion of theoligonucleotide, and N-mer denotes a random N-mer priming sequence usedto prime the template nucleic acids. See, e.g., U.S. Pat. ApplicationNo. 14/316,383, filed June 26. 2014, the full disclosure of which ishereby incorporated herein by reference in its entirety for allpurposes.

Following bead dissolution, the droplets are thermocycled to allow forprimer extension of the barcode oligos against the template of thesample nucleic acids within each droplet. Thermocycling results ingeneration of barcoded copy fragments of the sample nucleic acids thatinclude the barcode sequence representative of the originatingpartition, in addition to the other included sequences set forth above.Such an amplification reaction is described in more detail elsewhereherein.

After generation of the barcoded copy fragments, the emulsion ofdroplets including the barcoded copy fragments are destabilized with theaddition of a perfluorooctanol (PFO). This results in a pooled mixturecontaining the contents of the droplets. The pooled mixture is contactedwith Dynabeads MyOne Silane Beads (Life Technologies) in the presence ofguanidine thiocyanate (GuSCN) to promote the binding of the barcodedcopy fragments to the Dynabeads. The vessel containing the pooledmixture is placed into the holder of a magnetic device such that theDynabeads are pulled to the wall of the vessel. The supernatant isremoved and the Dynabeads are washed with 70% ethanol and vortexed tomix. The device is then flipped over and the vessel containing thepooled mixture is placed into the holder of the magnetic device suchthat the Dynabeads are pulled to the bottom of the vessel. Thesupernatant (washing agent) is removed. An elution agent is added to theDynabeads and the mixture is heated to 65° C. to elute the amplifiedfragments off of the Dynabeads. The eluted barcoded copy fragments arecollected, further processed to add any additional functional sequencesdesired and/or necessary for Illumina sequencing and sequenced on anIllumina sequencer.

In some cases, prior to further processing, the eluted barcoded copyfragments are collected and further purified by contacting the elutedbarcoded copy fragments with SPRI beads (Beckman-Coulter) in thepresence of poly ethylene glycol (PEG) such that the eluted barcodedcopy fragments bind to the SPRI beads. The SPRI beads undergo anotherround of separating (magnetic), washing, separating, and eluting toprovide further purified barcoded copy fragments. Following furtherpurification the further purified barcoded copy fragments can be furtherprocessed to add any additional sequences desired and/or necessary forIllumina sequencing and sequenced on an Illumina sequencer.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is: 1-34. (canceled)
 35. A method for purifying a target molecule, comprising: a) in a vessel, providing a support to a liquid mixture comprising contents of a destabilized droplet that comprise a target molecule, wherein the support binds the target molecule to provide a bound target molecule; b) immobilizing the support at a first location of the vessel, thereby separating the support from the liquid mixture; c) removing the liquid mixture from the vessel; d) providing a suspension fluid to the vessel, thereby suspending the support in the suspension fluid; and e) immobilizing the support at a second location of the vessel, thereby separating the support from the suspension fluid, wherein the second location of the vessel is different than the first location of the vessel.
 36. The method of claim 35, wherein, in a), the liquid mixture further comprises a chaotrope that aids in the support binding the target molecule.
 37. The method of claim 36, wherein the chaotrope comprises guanadine thiocvanate or guanidine hydrochloride.
 38. The method of claim 35, wherein, in a), the liquid mixture further comprises a destabilization agent capable of destabilizing an emulsion.
 39. The method of claim 38, wherein the destabilization agent comprises perfluorooctanol.
 40. The method of claim 35, wherein b) further comprises magnetically immobilizing the support at the first location of the vessel.
 41. The method of claim 35, wherein c) further comprises removing the liquid mixture from the vessel via suction.
 42. The method of claim 35, wherein c) further comprises removing the liquid mixture from the vessel via decanting.
 43. The method of claim 35, wherein e) further comprises magnetically immobilizing the support at the second location of the vessel.
 44. The method of claim 35, further comprising, after e), releasing the bound target molecule from the support, to provide a released target molecule.
 45. The method of claim 44, prior to the releasing the bound target molecule from the support, washing the support in one or more wash cycles by contacting the support with a washing agent.
 46. The method of claim 44, further comprising, after the releasing the bound target molecule from the support, subjecting the released target molecule to a solid phase reversible immobilization process.
 47. The method of claim 35, wherein the target molecule comprises a target nucleic acid molecule.
 48. The method of claim 47, further comprising, after e), determining a sequence of the target nucleic acid molecule.
 49. The method of claim 35, further comprising, after e), appending one or more additional nucleotides to the target nucleic acid molecule to provide a larger target nucleic acid molecule.
 50. The method of claim 49, further comprising determining a sequence of the larger target nucleic acid molecule.
 51. The method of claim 35, wherein the target molecule comprises a small molecule, a protein, or a peptide.
 52. The method of claim 35, wherein the support comprises a magnetic material or a particle.
 53. The method of claim 35, wherein the support is functionalized with a silanol or carboxylate that aids the support in binding the target molecule.
 54. A method for recovering target molecules from a plurality of droplets, comprising: a) providing a plurality of droplets having contents that comprise a plurality of target molecules; b) releasing the contents from the plurality of droplets to provide released target molecules; c) contacting the released target molecules with one or more supports in the presence of a chaotrope, wherein the one or more supports bind the released target molecules to provide bound target molecules, and wherein the one or more supports comprises at least one component responsive to a magnetic force; and d) applying the magnetic force to the one or more supports. 